Revision as of 19:21, 20 December 2010

Materials

preparing 10 ml cultures with 1, 10, 100, and 1000 μl of infected cultures assures that one of these will be ready the next day at the correct OD level. Select cultures which are just changing color.

chilled mycoplasma electroporation buffer

8 mM HEPES pH 7.4

272 mM sucrose (90.1 g/l)

chilled 1 mm electroporation cuvettes

10 ml chilled ATCC 1161 medium

Selective medium or plates

Tetracycline resistance from TetM is higher than 200 μg/ml

Untransformed cells grow poorly at 4 μg/ml

Tet selective plates are at 15 μg/ml

Protocol

chill the centrifuge to 4°

spin down 10 ml of overnight culture at 8000g for 5 minutes, remove the supernatent

resuspend the pellet in the remaining liquid by vigorous vortexing

add 10 ml of chilled electroporation buffer and mix

spin down again, remove supernatent

resuspend the pellet in the remaining liquid

add 10 ml of chilled electroporation buffer

spin down again, remove supernatent

resuspend the pellet in the remaining liquid, bring the total volume to 1200 μl with EPB

Freeze 400 μl aliquots at -80 indefinitely or use immediately

Add 16 μl of transposome or plasmid DNA for transformation

Mix and transfer to four chilled 1 mm gap electroporation cuvettes

Pulse the cuvette at 1.5 KV

Add ml of chilled 1161 medium immediately, mix with the pipet, cover

Place the cuvettes into the 30° incubator for outgrowth for 50 minutes

cells can be held at 4° following outgrowth

dilute 1 μl of the culture into 1 ml of 1161 medium, vortex, and dilute again into 1 ml. Plate 200 μl on a nonselective 1161 plate for counting untransformed CFUs.

Plate 360 μl aliquots on three selective plates

grow plates for 1.5 - 2 days at 30° for colonies

Notes

9/6/07: Arcing at 1.8 and 1.6 KV; possibly we need a second wash and/or some additional delay in the wash to remove more salt.
Gel loading tips do not work. Plating 125 ul seems to work well. Tested outgrowth at 2 ug/ml, 4 ug/ml, 8 ug/ml